Nitric oxide (NO) modulates cardiac muscle contraction via multiple pathways. Our long term objective is to understand the mechanisms by which NO and/or nitric oxides (NOx) alter cardiac contractility. We hypothesize that NO changes cardiac contractility by altering Ca2+ handling and the myofilament response to Ca2+ through both through cGMP-dependent and cGMP- independent mechanisms. During different stages of development of hypertrophy we will determine: 1) expression and activity of iNOS and eNOS in myocytes and whole heart and correlate these changes with alterations in contractility; 2) NO concentration in plasma; 3) mechanisms responsible for alterations in Ca2+ fluxes and myofilament response to Ca2+ by NO (NOx); and 4) the role of NO (NOx) in the altered response to adrenergic stimulation. We hypothesize that: 1) altered contractility by NO (NOx) is not only due to changes in Ca2+ current, but also due to changes in phosphorylation of phospholamban (PLB) and cardiac troponin I (cTnI), and 2) NO produced by iNOS plays a crucial role in the altered response to beta-adrenergic stimulation during different stages of development of hypertrophy. The studies employ hearts from control, iNOS knockout, and transgenic (TG) animals in which pressure overload is introduced by aortic banding, producing different phases of hypertrophy. The following TG mice are used: 1) TG mice which are deficient in PLB and either express native cTnl, or slow skeletal troponin I (ssTni) lacking phosphorylation sites for PKA and 2) TG mice that express normal level of PLB and either express cTnI or ssTnl. These TG mice allow us to separate the effect of alteration of TnI phosphorylation from PLB phosphorylation by NO (NOx). Our experiments provide new insights into the mechanisms and importance of NO (NOx) in alteration of cardiac contractility in physiological and pathological conditions.